(1) Field of the Invention
The present invention relates to the degradation of mixtures of polychlorinated biphenyls (PCBs) which occur as contaminants in the environment using a white rot fungus, Phanerochaete chrysosporium. In particular, the present invention relates to the degradation of mixtures of PCBs which contain up to about 60% by weight of chlorine and an average of 3 to 6 chlorine atoms per biphenyl molecule in contaminated compositions, particularly soil.
(2) Description of Related Art
Polychlorinated biphenyls (PCBs) are a family of compounds with a wide range of industrial applications in heat transfer fluids, dielectric fluids, hydraulic fluids, flame retardants, plasticizers, solvent extenders, and organic diluents. In the United States and the United Kingdom, complex PCB mixtures were manufactured under the trade name AROCLOR; among these are AROCLORS 1242, 1254, and 1260, which contain 42, 54 and 60% chlorine by weight (the last two digits represent the percent chlorine by weight), with an average of 3, 5 and 6 chlorines per biphenyl molecule, respectively. Aroclors consist of many congeners which differ in the number and distribution of chlorines on the biphenyl nucleus. About 150 congeners have been reported in the environment. PCBs have entered into soil and sediment environments as a result of improper disposal of industrial PCB wastes and leakage of PCBs from electric transformers. Their chemical inertness, due to a stable molecular structure and hydrophobicity, is believed to be responsible for their low biodegradation in ecosystems, leading to their persistence in the environment.
Some PCB congeners have been shown to be transformed by both aerobic and anaerobic bacteria (Abramowicz, D. A., Crit. Rev. Biotechnol. 10:241-251 (1990); and Quensen, J. F., III, et al., Appl. Environ. Microbiol. 56:2360-2369 (1990)). The aerobic biodegradation of PCBs is generally limited to less-chlorinated congeners (5 or fewer chlorines per biphenyl molecule) by a mechanism involving deoxygenase attack of the aromatic ring (Bedard, D. L., et al., Appl. Environ. Microbiol. 53:1094-1102 (1987); and Boyle, A. W, et al., Biodegradation 3:285-298 (1992)). The more-chlorinated congeners are generally recalcitrant to aerobic degradation. For instance, the aerobic microbial degradation of most of the congeners of Aroclor 1242 and that of some of the congeners of Aroclor 1254 have been demonstrated in the past, but there has been no convincing evidence to date for aerobic microbial degradation of Aroclor 1260. In contrast to aerobic biodegradation, PCBs undergo reductive dechlorination under anaerobic conditions, leading to the transformation of the more-chlorinated congeners to less-chlorinated congeners usually leaving the biphenyl ring intact. Chlorines substituted in the ortho position are generally recalcitrant to anaerobic dechlorination (GE Progress Report, General Electric Company Research and Development program for the destruction of PCBs: twelfth progress report for the period Aug. 1, 1992-July 31, pages 117-127 & 129-141 1993. General Electric Co. Corporate Research and Development, Schenectady, N.Y. (1993); and Quensen, J. F., et al, Appl. Environ. Microbiol. 56:2360-2369 (1990)).
Phanerochaete chrysosporium, a lignin-degrading white rot fungus, is known to mineralize a wide range of chloroaromatic environmental pollutants to CO.sub.2 (Bumpus, J. A., et al., Bioassays 6:116-120 (1987); Reddy, C. A., Current Opinion in Biotechnology 6:320-328 (1995); and Hammel, K. E., Oxidation Of Aromatic Pollutants By Lignin-degrading Fungi and Their Extracellular Peroxidases, p. 41-60. In H. Sigel and A. Sigel (ed.), Metal ions in biological systems, vol. 28. Degradation Of Environmental Pollutants By Microorganisms And Their Metalloenzymes. Marcel Dekker, Inc., New York (1992)). Degradation of many of these pollutants was reported to be mediated by the lignin-degrading enzyme system of this organism. Major components of the lignin-degrading enzyme system include lignin peroxidases (LIPs), Mn (II)-dependent peroxidases (MNPs), and the H.sub.2 O.sub.2 -producing enzyme system, which are induced during secondary metabolism, under nutrient-limiting culture conditions, but not under nutrient-rich conditions (Reddy, C. A., J. Biotechnol. 30:91-107 (1993)). More recently, a number of chloroaromatic pollutants were shown to be degraded by P. chrysosporium under nonligninolytic culture conditions (such as in defined high-N medium or nutrient-rich malt extract medium) when LIPs and MNPs are not produced (Kohler, A., et al., Appl. Microbiol. Biotechnol. 29:618-620 (1988); Yadav, J. S., et al., Biotechnol. Lett. 14:1089-1092 (1992); Yadav, J. S., et al., Appl. Environ. Microbiol. 59:2904-2908 (1993); and Yadav, J. S., et al., Appl. Environ. Microbiol. 61:677-680 (1995)).
Previous studies (Bumpus, J. A., et al., Science 228:1434-1436 (1985); and Thomas, D. R., et al., Biotechnol. Bioeng. 40:1395-1402 (1992)) demonstrated low levels of mineralization of 0.9 to 1.1% for individual PCB congeners, such as 3,3',4,4'-chlorobiphenyl (CB), 2,2',4,4'-CB, and 2,2',4,4',5,5'-CB when these were added to cultures at a low concentration (0.036 to 1.55 ppm) under ligninolytic culture conditions, i.e., at 2.4 mM ammonia and 56 mM glucose. Eaton (Eaton, D. C., Enzyme Microb. Technol. 7:194-196 (1985)) reported .ltoreq.9% mineralization when 0.25 ppm of Aroclor 1254 was added to such cultures. Although these data suggested mineralization of selected PCBs and PCB mixtures of P. chrysosporium , some skepticism of the reported PCB-degrading potential of this organism was expressed because of the low concentrations tested (Abramowicz, D. A., Crit. Rev. Biotechnol. 10:241-251 (1990)). There are no convincing data on the ability of the fungus to show substantial degradation of PCB mixtures at environmentally relevant concentrations or at levels degraded by known bacterial systems. Such data are needed to evaluate the PCB bioremediation potential of P. chrysosporium. Furthermore, relative degradation of individual PCB congeners in different Aroclor mixtures is not known. Such information is useful for comparing the nature and extent of degradation of various PCB congeners in fungal versus bacterial systems (Abramowicz, D. A., Crit. Rev. Biotechnol. 10:241-251 (1990)). Also, degradation of the highly chlorinated PCB mixture Aroclor 1260 has not been demonstrated before in either bacterial or fungal systems (Abramowicz, D. A., Crit. Rev. Biotechnol. 10:241-251 (1990)). Also, PCB mixture degradation by P. chrysosporium under non-ligninolytic conditions--(i.e. under nitrogen-rich and carbon-rich conditions when LIPs and MNPs are not produced) has not been demonstrated. This is important because nutrient-rich conditions prevail at some contamination sites.